Abstract The incidence of heart failure (HF) continues to increase in the Western world and HF related morbidity and mortality remain unacceptably high. Advances in understanding the molecular mechanisms that are associated with cardiac failure have offered a number of important targets for intervention. Two critical abnormalities in failing cardiomyocytes are: 1) an abnormal sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) pump function and 2) decreased expression of SERCA2a. The importance of reduced SERCA2a expression in HF is documented in many studies utilizing experimental animal models and restoration of SERCA2a by gene transfer significantly rescues HF phenotype and increases animal survival. Our group has targeted the reduced expression of SERCA2a by delivering intracoronary adeno-associated vector type 1 encoding SERCA2a (AAV1.SERCA2a) in patients with heart failure. During the previous funding period, our group found that post- translational modifications (PTMs) of SERCA2a can significantly affect the function of this pump. We discovered that the levels and activity of SERCA2a in cardiomyocytes are modulated by small ubiquitin-like modifiers type 1 (SUMO-1). Reduced SUMO-1 levels and decreased SERCA2a SUMOylation were found in failing human myocardium. In the first four years of our proposal, we showed that in murine studies, AAV- mediated SUMO-1 gene delivery significantly improved SERCA2a's levels, cardiac function and increased mouse survival in pressure overload-induced HF. We also found that SUMO-1 gene transfer improved contractility and prevented left ventricular dilation in failing pig hearts. SUMOylation was found to be a critical PTM that regulates SERCA2a function. There is also growing evidence that aside from SUMOylation other PTMs such as oxidation, methylation, nitration, phosphorylation and acetylation occur in cardiac cells. The crosstalk and selectivity of different lysine PTMs of SERCA2a could be an important mechanism for cells to respond to different stimuli in a time-dependent fashion. Our recent findings show that SERCA2a SUMOylation is closely linked to lysine acetylation/deacetylation pathways. First, SERCA2a SUMOylation is decreased in HF while acetylation is increased in the setting of HF. Second, SERCA2a acetylation negatively impacts SERCA2a's function. Third, our preliminary data show that the reduction of acetylation is accompanied by increased SUMOylation of SERCA2a. These data suggest that there is a balance between acetylation and SUMOylation of SERCA2a, which is altered in HF. The major hypothesis of our proposal is that SERCA2a acetylation decreases SERCA2a function and reduces SUMOylation of SERCA2a leading to worsening cardiac function in the setting of HF. Achieving these aims will provide new insights into the mechanisms of lysine-mediated SERCA2a regulation in the setting of HF. This study will provide a novel strategy for manipulating SERCA2a post-translational modifications that may ultimately lead to novel therapeutic agents.